Stempro adipogenesis differentiation media

Adipogenic differentiation was performed using StemPro Adipogenesis differentiation media (Invitrogen, Carlsbad, CA, USA) for 14 days. After that, cells were washed twice with PBS, fixed in 4% formaldehyde for 15 min and stained with oil red O stain, to confirm the presence of adipocytes. StemPro Osteogenic differentiation kit (Invitrogen) was used to induce ADSCs differentiation towards the osteogenic lineage. After 21 days in culture, differentiated cells were washed, fixed in 4% formaldehyde for 15 min, and stained with Alizarin Red S (ARS) stain, to verify the osteogenic differentiation. Cells under normal culture conditions were used as negative controls.

For colony forming unit-fibroblast (CFU-F) assays, MSCs were plated 1000 cells in 100mm tissue culture dishes. MSCs and iMSCs were maintained in DMEM containing 15% FBS, L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified 5% CO2 at 37 °C. 14 days after plating, the cells were fixed with methanol and then stained with crystal violet (Sigma) for visualization.
Osteogenic differentiation was induced by StemPro osteogenesis differentiation media (Thermo Fisher). MSCs or iMSCs exchanged differentiation medium every 3 days. After 8 days, the mineralization of the extracellular matrix was determined by Alizarin Red S (Sigma) staining, and extraction with 10% cetylpyridinium chloride (Sigma) followed by measurement of the absorbance at 405 nm.
For adipogenic differentiation, confluent cells were incubated in StemPro adipogenesis differentiation media (Thermo Fisher). MSCs or iMSCs exchanged differentiation medium every 3 days. After 10 days, cells were fixed with propylene glycol (Sigma) and stained with Oil Red O (Sigma) to visualize lipid droplets. The numbers of lipid droplets were subsequently determined by dye extraction using 4% Nonidet P40 (Sigma) in isopropyl alcohol (Sigma) followed by spectrophotometry at 520 nm.